An integrated liquid chromatography–tandem mass spectrometry approach for the ultra-sensitive determination of catecholamines in human peripheral blood mononuclear cells to assess neural-immune communication

Abstract

Catecholamines play a vital role in the interactions between the nervous and immune systems and their dysfunctions are implicated in various autoimmune and neurological diseases. However, accurate quantitation of catecholamines in the immune system presents a special analytical challenge. We proposed the first LC–MS/MS method for the determination of catecholamines in human peripheral blood mononuclear cells (PBMC) with significantly improved sensitivity, selectivity and throughput without requiring derivatization, evaporation and ion-pairing reagent. PBMC were separated by density gradient centrifugation and lysed with 0.2M acetic acid. The analytical novelty includes the first solid phase extraction on a 96-well hydrophilic-lipophilic-balanced (HLB) μElution plate upon complexation with phenylboronic acid (PBA), enabling specific clean-up and fivefold pre-concentration of catecholamines in a single extraction. LC chromatographic separation was obtained on a PFP column with 0.01% HCOOH as additive with enhanced signal response. Summation of five MRM transitions yielded three–four fold rise in sensitivity. The lower limit of quantification of 1pg/mL for epinephrine (E) and 5pg/mL for norepinephrine (NE) and dopamine (DA) represents a considerable sensitivity improvement over available methods. Less than 8.7% of intraday and interday precision, 91.8–111.3% of accuracy and successful assessment of reference intervals for 40 healthy donors suggested good reproducibility and reliability of the assay. The novel PBA-HLB-PFP-MRM summation approach allows rapid, sensitive and reliable determination of catecholamines in PBMC, which will facilitate better understanding of the new arena of neural-immune network. Additionally, the substantially improved method can be modified to quantify catecholamines and metabolites in other biological matrices.